Data safety and monitoring board(DSMB) have been performed when half of sample size (SS)have been reached. Recruitment was slower than expected so SS was recalculated. After analysis of data, DSMB recommended to stop study.

Background: Despite management improvement in lasts years, S.aureus bacteremia leads to high morbidity and mortality. For over 50 years, methicillin-susceptible S.aureus (MSSA) bacteremia standard treatment was cloxacillin. Previous studies using different therapies and combination treatment fall to improve survival in these patients.

Aim: to demonstrate the efficacy of the cloxacillin and fosfomycin combination administered during the first week of treatment, compared with cloxacillin monotherapy in patients with MSSA bacteremia in treatment success. Methods: A multicentre, superiority, open-label, randomized, phase IV-III, two-armed parallel (1:1) groups clinical trial. Adult patients with MSSA bacteremia will be randomized to Combination therapy group: patients will receive intravenous cloxacillin 2g/4h and fosfomycin 3 g/6h for the duration of 7 days treatment, or Standard therapy group: patients will receive intravenous cloxacillin 2g/4h for the duration of 7 days IV treatment. After the first week, antibiotic treatment and duration will be decided by responsible clinician following clinical practice.

The primary endpoint is the treatment success measured at day 7 of treatment; a composite endpoint defined by all of the following criteria met after randomization: patient alive at day 7 AND stable or improved quick SOFA score (compared with baseline) at day 7 AND fever resolved at day 7 AND negative blood cultures for S. aureus at day 7.

In case of achieving statistical differences in the primary endpoint, investigators will perform a hierarchical analysis of the treatment success at Test of Cure visit (TOC, 12 weeks after randomization), defined by the presence of all of the following: patient alive at TOC AND no evidence of microbiological treatment failure defined as isolation of S. aureus from blood culture or other sterile site from day 8 after randomization until TOC.

Investigators have assumed a 74% of treatment success in monotherapy group. Accepting an alpha risk of 0.05 and a beta risk of 0.2 in a two-sided test, 183 subjects are necessary in first group and 183 in the second to find a statistically significant difference of 12%. It has been anticipated a drop-out rate of 5%.

Discussion: Randomized studies assessing efficacy of different treatment in MSSA bacteremia are lacking. This study could help to improve knowledge about MSSA bacteremia and whether combined treatment with cloxacillin and fosfomycin could improve outcomes compared with standard treatment.

SAFO trial is a multicentre, superiority, open-label, randomized, phase IV-III, two-armed parallel (1:1) groups clinical trial comparing combination treatment with fosfomycin and cloxacillin with standard therapy with cloxacillin in adult patients with MSSA bacteremia.

Patients will be randomized to:

• Standard treatment group: patients will receive intravenous cloxacillin 2g/4h for the duration of 7 days IV treatment. If creatinine clearance is <30 mL/min cloxacillin will be administrated at dose of 2g every 6 hours.
• Combination therapy group: patients will receive intravenous cloxacillin as explained above and fosfomycin 3 g/6h for the duration of 7 days treatment. In case of renal failure, fosfomycin will be administrated as follow:

Creatinine clearance (mL/min) Fosfomycin dosage >40 3 g every 6 hours 20-40 3 g every 12 hours 10-20 3g every 24 hours <10 3 g every 48 hours Haemodialysis 3 g after haemodialysis Continuous renal replacement therapy 3 g every 24h hours

The duration of overall antibiotic treatment and the duration of intravenous treatment will be determined according to clinical criteria depending on status (complicated or uncomplicated bacteremia, source of infection) by responsible clinician according with current guidelines. Patient with complicated bacteremia will receive at least 4-6 week of antibiotic treatment.

Primary endpoint

Treatment success at day 7 is a composite outcome defined by all of the following criteria met after randomization:

• Patient alive at day 7 AND
• Clinical improvement measured by stable or improved quick SOFA score (compared with baseline) at day 7 AND
• Fever resolved at day 7 AND
• Negative blood cultures for S. aureus at day 7.

In case of statistical differences observed between groups in the primary endpoint, investigators will perform a hierarchical testing analyzing the treatment success at Test of Cure (TOC visit, 12 weeks after randomization).

Treatment success at TOC visit is defined by presence of all of the following:

• Patient alive at TOC;
• No isolation of MSSA in blood culture or in another sterile site from day 8 until Test of Cure visit (TOC, 12 weeks after randomisation). In case of patients with prolonged course of antibiotic treatment (more than 10 weeks), TOC visit will be performed two weeks after the end of treatment (EOT).

Treatment failure is defined by the presence of one of the following condition: all-cause mortality at TOC, positive blood cultures at day 7 or later, withdraw of the study because of adverse events related to study treatment, requirement of an additional MSSA-active antibiotic until day 7, lacking of clinical improvement at day 7.

Secondary endpoint

Clinical secondary endpoints:

• To compare all-cause mortality at days 7, 14, EOT and 90 after randomization in cloxacillin treatment group versus cloxacillin and fosfomycin treatment group.
• To evaluate persistent bacteremia (at least one positive blood culture) at day 3 and persistent bacteremia at day 7 after randomization in the two arms of treatment.
• To determine the microbiological relapse as defined by at least one positive blood culture for MSSA at least 72 hours after a preceding negative culture in the two arms of treatment.
• To evaluate microbiological treatment failure as defined by positive sterile site culture for MSSA at least 14 days after randomisation in the two arms.
• To determine the number of patients with persistent and relapsing bacteremia in the two arms of treatment.
• To evaluate the number of patients with complicated bacteremia, defined by persistent bacteremia, endocarditis or metastatic emboli, prosthetic devices) in the two arms of treatment.
• To determine the length of stay in intensive care unit and in hospital in both arms of treatment.
• Duration of intravenous antibiotic treatment. Sub group analysis for patients at high risk (persistent bacteraemia, metastatic infection, unknown focus of bacteraemia, endocarditis, pneumonia).

Microbiological secondary endpoints:

• To determine emergency of fosfomycin-resistant strains during therapy in the arm of combination treatment.
• To evaluate operon agr functionality and its relationship with Minimum Inhibitory Concentration (MIC) changes to vancomycin (VAN) and daptomycin (DAP) and with biofilm production.
• To analyze VAN and DAP MIC as markers of complications during bacteraemia.
• To determine the "in vitro" cloxacillin plus fosfomycin combination synergy.
• To realize whole genome sequencing and its changes in patients with treatment failure.

Pharmacological secondary endpoints:

• To determine minimum and maximum concentration in steady state of fosfomycin and cloxacillin.
• To evaluate pharmacokinetic variability of these concentration.
• To study the association between PK parameters and efficacy.

Security secondary endpoints:

To evaluate the security of cloxacillin and fosfomycin combination compared with cloxacillin monotherapy.

Inclusion Criteria:

• Subjects, aged ≥ 18 years;
• MSSA bacteraemia: ≥ 1 positive blood culture(s) for MSSA in the first 72 h up to randomisation in patients with clinical suspicion of infection;
• Written informed consent of the participant or the legal representative.

Exclusion Criteria:

• Severe clinical status with expected death <48h.
• Severe hepatic cirrhosis (Child-Pugh C).
• Moderate-severe cardiac chronic failure (NYHA III-IV).
• Prosthetic endocarditis (need for concomitant antibiotic therapy active against S. aureus together with the study antibiotics for the first 7 days of the study).
• No pre-existing evidence of S. aureus fosfomycin non-susceptibility.
• Known hypersensitivity to cloxacillin or fosfomycin.
• Polymicrobial bacteraemia with more than one microorganism in blood cultures.
• A positive pregnancy test or pregnancy or lactation at the time of inclusion.
• Miastenia gravis.
• Participation in another clinical trial.
• Previous participation in the present clinical trial.
• Acute SARS-CoV2 infection.

Sharing of data generated by this project is an essential part of our proposed activities and will be carried out in several different ways. We would wish to make our results available both to the community of scientists interested in infectious diseases and the biology of Staphylococcus aureus to avoid unintentional duplication of research.

The preliminary results will be presented at international and national infectious diseases conferences and will be published in peer-reviewed journals. The results will also be made available through press and social media communications. Any formal presentation or publication of data collected from this study will be considered as a joint publication by the participating investigators and will follow the recommendations of the ICMJE. Individual participant data that underlie the results, after deidentification will be available. Proposals should be directed to the corresponding author.

immediately following publication and ending 5 years following article publication

Data will be shared with researchers who provide a methodologically sound proposal to achieve aims in the approved proposal.